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There is increasing evidence that cyclooxygenase (COX)-2 possess both angiogenic and cardioprotective properties. We examined the effects of hypoxic cardiac myocytes (H9c2 cells) on COX-2 expression in human umbilical vein endothelial cells (HUVECs) to determine the pathway involved in COX-2 regulation. The medium from hypoxic ( cyclooxygenase-2; vascular endothelial growth factor; cardiac myocytes

Shie, Jue-Lon, Zhi Y. Chen, Michael J. O'Brien, Richard G. Pestell, Mu-En Lee, and Chi-Chuan Tseng. Role of gut-enriched Kruppel-like factor in colonic cell growth and differentiation. Am J Physiol Gastrointest Liver Physiol 279: G806-G814, 2000.--Cancer cells differ from normal cells in many aspects, including hyperproliferation and loss of differentiation. Recent research has focused on the role of transcription factors in regulating abnormal cell growth. Gutenriched Kruppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract. In the current study, we demonstrated that GKLF mRNA levels were significantly decreased in the dysplastic epithelium of the colon, including adenomatous polyp and cancer. GKLF immunostains in the normal colon were higher at the surface epithelium and gradually decreased toward the crypt, but this gradient was not present in the adenomatous and cancerous mucosa. Constitutive overexpression of GKLF DNA in a human colonic adenocarcinoma cell line (HT-29) decreased [[sup.3]H]thymidine incorporation, whereas suppression of GKLF gene increased DNA synthesis, indicating that downregulation of the GKLF gene might contribute to cellular hyperproliferation. Cyclin D1 (CD1) protein level and CD1-associated kinase activity were decreased in HT-29 cell overexpressed GKLF cDNA, and CD1 promoter activity was profoundly suppressed by GKLF. When HT-29 cells were cultured in the presence of sodium butyrate, GKLF mRNA levels increased as cells acquired more differentiated phenotypes. These results suggest that GKLF plays an important role in regulating cell growth and differentiation in the colonic epithelium and that downregulation of GKLF expression may cause colonic cells to become hyperproliferative. Furthermore, GKLF appears to be a transcriptional repressor of the CD1 gene. colon cancer; adenoma; proliferation; transcription factor

Gut-enriched Kru¨ppel-like factor (GKLF/KLF4), a member of the Kru¨ppel-like factor family of transcription factors, has recently been shown to be associated with growth arrest in the colonic mucosa. Our laboratory has previously demonstrated that GKLF is one of the down-stream targets of interferon-γ (IFN-γ) in colon cancer cells, but the signaling pathway regulating GKLF expression is not known. In this report, we showed that IFN-γ increased GKLF and interferon regulatory factor-1 mRNA levels in a parallel fashion and these effects were abolished in STAT1 knockout mouse fibroblast cell lines, indicating that STAT1 mediated IFN-γ-stimulated GKLF gene expression. IFN-γ treatment induced phosphorylation of STAT1 and IFN-γ-induced GKLF mRNA expression was attenuated by tyrosine protein kinase inhibitors, suggesting that phosphorylation of STAT1 played an essential role in this process. To further evaluate the effect of STAT1 on GKLF gene expression, a 2622-bp mouse GKLF promoter was isolated from a liver genomic library. In a transient transfection system, IFN-γ treatment increased GKLF promoter activity by 3.5-fold. Sequential deletion and mutation analysis of the GKLF promoter has identified the sequence between −1675 and −1580, a region containing a GAS element, to be essential for IFN-γ function. By electrophoretic mobility gel shift assay, nuclear extracts from IFN-γ-stimulated HT-29 cells were found to bind to the GAS motif on the GKLF promoter and this protein–DNA complex was supershifted by the STAT1 antiserum. These results indicate that IFN-γ-induced GKLF expression required phosphorylated STAT1 and that these effects were mediated, in part, through interaction of STAT1 with the GAS element on the GKLF promoter. [Copyright &y& Elsevier]

Abstract: Fibroblast growth factor receptor-1 (FGFR-1) has been implicated in the process of cardiogenesis, although the underlying molecular mechanisms are poorly understood. In this study, we report the regulation of FGFR-1 expression by related transcriptional enhancer factor-1 (RTEF-1) in vitro (endothelial cells) and in vivo (RTEF-1 transgenic mice). FGFR-1 promoter activity, FGFR-1 mRNA and protein level were measured in bovine aortic endothelial cells (BAEC) in response to RTEF-1 and in endothelial cells isolated from livers in RTEF-1 transgenic mice. RTEF-1 stimulated FGFR-1 promoter activity in a dose-dependent manner. RTEF-1 enhanced FGFR-1 mRNA (4-fold) and protein expression (3.5-fold) whereas RTEF-1 siRNA decreased FGFR-1 protein expression (4-fold). FGFR-1 mRNA and protein expression were also increased in endothelial cells isolated from livers of RTEF-1 transgenic mice. Furthermore, RTEF-1 enhanced tubule formation whereas this was decreased by RTEF-1 knockdown. Moreover, increased relaxation of microvessels was found in RTEF-1 transgenic mice compared to wild-type mice. Our results indicate that RTEF-1 acts as a transcriptional stimulator of FGFR-1 in endothelial cells through its activation of the FGFR-1 promoter. RTEF-1 thus plays an important role in the regulation of FGFR-1 expression. These findings help further understand FGFR activity in angiogenesis and may lead to new therapeutic targets in ischemic vascular disorders. [Copyright &y& Elsevier]